PROJECT SUMMARY Limb congenital defects are among the most common causes of disability in the United States affecting 0.8/1000 children born each year. Recent realizations of plausible regenerative medicine therapies in humans has increased efforts towards understanding examples of animal regeneration and the mechanisms that drive the process. In this proposal, we aim to provide insight into the mechanisms that facilitate regeneration by studying a clinically relevant signaling molecule, retinoic acid, during limb regeneration in the salamander. Axolotl salamanders (Ambystoma mexicanum) are particularly useful, because they regenerate entire limbs after amputation by generating a heterogeneous mass of lineage- restricted progenitors called a blastema at the amputation site. The blastema uses positional information retained in connective tissue cells to regenerate the proper limb regions. The goal of this proposal is to the endogenous role in this process and study how positional information is established and maintained in connective tissue cells, which is critical to our understanding of limb regeneration. We will use unbiased single cell RNAseq and spatial transcriptomic approaches to compare gene expression at single-cell resolution throughout the regeneration process. We will also determine the transcriptional targets of retinoic acid specifically in limb connective tissue cells that are undergoing reprogramming from distal to proximal cell identity. Lastly, the positional memory of connective tissue cells will be experimentally programmed in vivo to demonstrate how positional memory is maintained. In the end, the major outcome of this project will be to know how a salamander limb knows what limb structures to grow back in order to stimulate new regenerative approaches in mammalian systems.